Transgenic mouse expressing the human cyclooxygenase-2 gene and neuronal cell cultures derived therefrom

ABSTRACT

The present invention relates to the use of nimesulide and structurally related compounds in the prevention and/or treatment of neurodegenerative conditions. It is based, at least in part, on the discovery that nimesulide exhibits a neuroprotective effect against &amp;bgr.-amyloid induced cell death Without being bound to any particular theory, it appears that nimesulide inhibits a non-inflammatory mechanism of neurodegeneration

1. INTRODUCTION

[0001] The present invention relates to the use of nimesulide andstructurally related compounds in the prevention and/or treatment ofneurodegenerative conditions such as Alzheimer's Disease. It is based,at least in part, on the discovery that nimesulide, in effectiveconcentrations, inhibits cell death.

2. BACKGROUND OF THE INVENTION 2.1. Alzheimer's Disease

[0002] Sporadic Alzheimer's Disease is the major neurodegenerativedisease associated with aging, the risk of developing the disease risingexponentially between the ages of 65 and 85, doubling every five years.Histologically, the hallmarks of Alzheimer's Disease are the depositionof amyloid in senile plaques and in the walls of cerebral blood vessels;the presence of neurofibrillary tangles, and neurodegeneration. Theetiology of Alzheimer's Disease, however, is not well understood.Genetic factors have been proposed to play a role, including trisomy 21,mutations in the amyloid β-protein precursor (“APP”) gene, thepresenilin-1 (PS 1) and presenilin-2 (PS2) genes, and the presence ofthe apolipoprotein E type 4 allele (Younkin, 1995, Ann. Neurol.37:287-288; Lendon et al., 1997, JAM A 277:825). Several studies haveindicated that β-amyloid induces apoptosis in cultured neurons (Loo etal., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7951-7955; Li et al., 1996,Brain Res. 738:196-204). Such induction may involve the immediate earlygene proteins, c-jun and fos (Anderson et al., 1995, J. Neurochemistry65:1487-1498; Anderson et al., 1994, Experimental Neurology 125:286-295;Anderson et al., 1996, J. Neurosci. 16:1710-1719).

[0003] It has been proposed that apoptosis may be involved in thepathogenesis of Alzheimer's disease (Smale et al., 1995, Exp. Neurol.133:225-230; Anderson et al., 1996, J. Neurosci. 16:1710-1719; Andersonet al., 1994, Exp. Neurol. 125:286-295). Inflammatory mechanisms havealso been implicated; (Pasinetti, 1996, Neurobiol. Ageing 17:707-716)supportive of such a mechanism are the observations that acute phaseproteins are elevated in the serum of Alzheimer's Disease patients, andare deposited in amyloid plaques; activated microglial cells tend tolocalize in the vicinity of senile plaques, and complement componentshave been localized around dystrophic neurites and neurofibrillarytangles (Aisen and Davis, 1994, Am.J.Psychiatry 151:1105-1113).Antiinflammatory agents have been suggested as potential therapeuticagents (Aisen et al., 1996, Dementia 7:201-206; McGeer et al., 1996,Neurology 47: 425-432). Because they tend to have fewer adverse sideeffects, selective inhibitors of the enzyme cyclooxygenase-2 have beenadvanced as agents for treating such inflammation (InternationalPublication No. WO 94/13635 by Merck Frosst Canada Inc.). Prior to thepresent invention, however, it had not been believed that such agentscould be used to inhibit non-inflammatory aspects of neurodegenerationin the context of Alzheimer's Disease or otherwise.

2.2. Cvclooxvgenase-2

[0004] Cyclooxygenases (“COXs”) are enzymes that catalyze the formationof prostaglandin (“PG”)-H₂ from arachidonic acid (AA). PG-H₂ is furthermetabolized to physiologically active PGs (e.g., PG-D₂, PG-E₂ andPG-F_(2α)), prostacyclin (PG-I_(2α)) and thromboxanes. Specific PGs havediverse, often antagonistic effects on different tissues. For example,PG-I₂ and PG-E₂ are potent vasodilators that may contribute to theinflammatory response, whereas PG-F_(2α), is a vasoconstrictor.

[0005] There are two known COX isoforms, COX-1 and COX-2, which, thoughphysiologically distinct, are similar in amino acid sequence andenzymatic functions. COX-1 is constitutively expressed at differentlevels in different cell types. COX-2, however, is not constitutivelyexpressed, and is generally undetectable in normal peripheral tissues(Kujubu et al., 1991, J. Biol. Chem. 266:12866-12872; O'Banion et al.1992, Proc. Natl. Acad. Sci. U.S.A. 89:4888-4892). Rather, COX-2expression is inducible (for example, by mitogens) and COX-2 mRNA levelshave been observed to rise rapidly in response to inflammatory stimulisuch as interleukin-1β and lipopolysaccharide, and to decrease inresponse to glucocorticoids. When subjected to these same factors, COX-1mRNA levels remain substantially unchanged, suggesting that COX-2 is theisoform which mediates inflammation (Cao et al., 1995, Brain Res.697:187-196; O'Banion et al. 1992, Proc. Natl. Acad. Sci. U.S.A.89:4888-4892).

[0006] Recent evidence suggests that COX-2 may play a role in mechanismsof cell survival and cell adhesion in peripheral cells (Lu et al., 1996,Proc. Natl Acad. Sci. U.S.A. 92:7961-7965; Tsujii et al., 1995, Cell83:493-501). Tsujii et al. reports that epithelial cells engineered toexpress elevated levels of COX-2 were resistant to butyrate-inducedapoptosis, exhibited elevated BCL2 protein expression, and reducedtransforming growth factor β2 receptor levels (Tsuji et al., 1995, Cell83:493-501). Lu et al. indicates that non-steroidal antiinflammatorydrugs may induce an apoptotic mechanism involving the COX system.

[0007] The roles of COX-1, COX-2 and PG synthesis in normal brain, andin the context of Alzheimer's Disease, have not been fully characterizedto date. The importance of PGs in brain physiology may be independent ofinflammatory mechanisms. In the brain, PG receptors have been identifiedin the hypothalamus, thalamus, and limbic system (Watanabe et al., 1989,Brain Res. 478:143-148). PGs are involved in hypothalamic-pituitaryhormone secretion (Kinoshita et al., 1982, Endocrinol. 110:2207-2209),regulation of temperature and the sleep-wake cycle (Hayaishi, 1988, J.Biol. Chem. 263:14593-14596). There is recent evidence that COX-2 mRNAis expressed and regulated in rat brain by synaptic activity andglucocorticoids (Adams et al., 1996, J. Neurochem. 66:6-13; Kaufmann etal., Proc. Natl. Acad. Sci. U.S.A. 93:2317-2321; Yamagata et al., 1993,Neuron 11:371-386). These studies indicate that COX-2 is regulated as animmediate early gene in the brain, and suggest that PGs may be importantin trans-synaptic signaling and long-term potentiation. Chang et al.(1996, Neurobiol. of Aging 17:801-808) report that COX-2 mRNA expressionis decreased in Alzheimer's disease.

3. SUMMARY OF THE INVENTION

[0008] The present invention relates to the use of nimesulide andstructurally related compounds in the prevention and/or treatment ofneurodegenerative conditions. It is based, at least in part, on thediscoveries that (i) COX-2 expression in models of neurodegeneration isincreased in neurons rather than glial cells (consistent with anon-inflammatory mechanism), and (ii) nimesulide exhibits aneuroprotective effect against β-amyloid induced neuronal cell death.This latter finding is particularly unexpected in view of the ability ofCOX-inhibitors to increase apoptosis of non-neuronal cells. Withoutbeing bound to any particular theory, it appears that nimesulideinhibits a non-inflammatory mechanism of neurodegeneration.

4. DESCRIPTION OF THE FIGURES

[0009]FIG. 1. Regional expression of COX-2 mRNA in control unlesionedmale rat brain. COX-2 mRNA expression was assessed by in situhybridization and visualized by X-ray autoradiography. Abbreviations:DG, dentate gyrus; CA1, CA2 and CA3 subregions of the neuronal pyramidallayer of the hippocampal formation; PAC, parietal cortex; PYC, pyriformcortex; AC, amygdaloid complex; THAL, ventroposterior thalamic nucleic.Adapted from plate 24 of Paxinos and Watson, 1986 (The Rat Brain inSteriotaxic Coordinates, Academic Press, NY).

[0010]FIG. 2. Maturational regulation of COX-2 mRNA expression in ratbrain. Optical densities were quantified from autoradiographic images.Abbreviations, as above and as follows: PYC/AC, pyriform and amygdaloidcomplex that were quantified for COX-2 mRNA expression as a single brainregion. N=5-8 per time point.

[0011]FIG. 3. Maturational influence on COX-2 mRNA expression duringresponse to KA-induced seizures. Micrographs were generated fromautoradiographic images. For anatomical distribution of changes, referto FIG. 1. Control, unlesioned vehicle-injected rats; KA 8 H, KA-treatedrats 8 hours prior to sacrifice; postnatal days P-7, P-14, and P-21.

[0012] FIGS. 4A-D. Time course of COX-2 mRNA changes in rat brain duringresponses to KA treatment: maturational influences and regionaldistribution of changes. Optical densities were quantified fromautoradiographic images. Data are expressed as means±SEM, N=4=6 pergroup. *P<0.0l vs 0 H group (saline injected group); 4 h, 8 h, 16 h, 30h, 120 h, time in hours after onset of KA-induced seizures.

[0013] FIGS. 5A-D. Maturational influence on the distribution of COX-2mRNA expression and induction in rat hippocampus. COX-2 mRNA in P21(A,B) and adult (C,D) hippocampal formations as assessed by in situhybridization assay and visualized by emulsion autoradiography usingdark-field illumination. In A and C, COX-2 mRNA expression in controlrats (vehicle injected); in B and D, COX-2 mRNA 8 hours after onset ofKA-induced seizures. Arrows point toward the superficial layer of the DG(stratum granulosum). Scale bar=200 μm.

[0014]FIG. 6. Selective induction of hippocampal COX-2 but not COX-1mRNAs during response to KA-induced seizures as assessed by gel blothybridization assay. CTL, control saline-injected rats; KA, KA-treatedrats 12 hours post lesioning.

[0015] FIGS. 7A-I. KA-induced COX-2 and apoptosis in adult rat brain. In(A,B), COX-2 mRNA expression in CA3 hippocampal pyramidal neurons ofcontrol and KA-treated rat, respectively; in (C), CA3 subregion of thehippocampal formation of KA-lesioned rats showing neurons with apoptoticfeatures (arrows). In (D,E), COX-2 mRNA expression in pyriform cortex ofcontrol and KA-treated rat respectively. In (F), arrows point towardapoptotic cells of pyriform cortex of KA-lesioned rats. In (G,H), COX-2mRNA expression in cells of the amygdaloid complex of control andKA-treated rats, respectively. In (I), arrows point toward apoptoticcells of the amygdaloid complex of KA-lesioned rats. COX-2 mRNA wasassayed by in situ hybridization and visualized by emulsionautoradiography under dark field illumination. In situ 3′ end-labelingwas used to assess apoptotic features following KA treatment. Scale bar:in A,B,D,E,G,and H=200 μm; in C,F and I=40 μm.

[0016] FIGS. 8A-D. Immunocytochemical evidence of neuronal COX-2expression/regulation in response to glutamate in vitro. COX-2-likeimmunoreactivity in monotypic cultures of rat primary hippocampalneurons in (A) control and (B) after glutamate exposure (12 hours). In(C,D) control and glutamate treated cultures immunoreacted withimmunoadsorbed COX-2 antibody, respectively. Scale bar=50 μm.

[0017] FIGS. 9A-C. Effect of nimesulide on endotoxin-mediatedsynthesis/secretion of cytokines and nitrites in glia. The effect of10⁻⁹ M nimesulide on the synthesis/secretion of (A) TNF, (B) NOintermediates (Griess reaction) and (C) PGE₂, as assessed in BV2 mouseimmortalized microglial cells. Mean±SEM, n=8-10 per group.Lipopolysaccharide (“LPS”)=5 μg/ml. LPS and nimesulide were added incombination to cultures; incubation time was 24 hours. Statistics usedANOVA, p<0.05.

[0018] FIGS. 10A-E. Time course of changes of COX-2 protein in P19 cellsduring response to conditions leading to apoptotic death. In (A),quantitative analysis of COX-2 induction in P19 cells, n=4-6 per group,p<0.05 vs. t=0. In (B), changes were assessed by western analysis, usingchemiluminescent detection. In (C,D) the morphological appearance ofapoptotic nuclei in P19 cells was assessed by Hoechst H33258 24 hoursafter serum removal (and replacement with N2 medium). In (E), theelectrophoretic profile of DNA showing DNA laddering degradation wasassessed 14 hours after serum removal (lane 1, control cells at t=0;lane 2, DNA laddering 14 hours after serum removal; lane 3, DNAmarkers). The COX-2 polyclonal antibody used in these studies was asdescribed in Section 6. The COX-2 specific antibody recognizes two majorbands having estimated molecular weights of about 70 and 65 kDa in totalhomogenates of mouse, rat and human brains.

[0019] FIGS. 11A-E. Regulation of COX-2 during response to Aβ1-40mediated oxidative stress in SH-SY5Y neuronal cells. (A) Bar graphshowing the amount of COX-2 immunoreactivity present relative to control(“CTL”). The immunoreactivity measurement was derived from the datashown in (B). (B) Western blot of proteins prepared from control SH-SY5Ycells or cells exposed to Aβ peptides for 48 or 72 hours, reacted withCOX-2 antisera. (C) Results of MTT assays of control or Aβ-treatedSH-SY5Y cells. (D) The immunoblot shown in (B) stripped andimmunoreacted with actin antisera. (E) SH-SY5Y cultures were examinedfor apoptotic mechanisms.

[0020]FIG. 12. Protective effect of nimesulide on Aβ1-40 mediatedneurotoxicity as assessed by MTT assay using SH-SY5Y neuronal cells.Abbreviations: CTL control; NIM nimesulide; A B-β-amyloid (1-40).*P<0.01 vs CTL, **P<0.05 vs. CTL.

[0021]FIG. 13. Micrographs showing induction of COX-2 immunoreactivityin temporal cortex of AD and age-matched neurological controls. Themicrographs show a selective induction of COX-2 immunostaining inneuron-rich grey matter (“gm”) but not glia-rich white matter (“wm”)regions.

[0022] FIGS. 14A-D. COX-2 immunostaining of neurons in frontal cortex ofAD brain and co-localization of COX-2 immunostaining with AD plaques infrontal cortex of AD brains. In (A,B), immunostaining of COX-2 neuronsof AD are shown (A, low power; B, high power magnification). In (C),immunostaining of diffuse plaques. In (D), COX-2 in neuritic plaques asassessed by Aβ immunostaining on adjacent tissue sections.

[0023] FIGS. 15A-D. COX-2 expression in AD brain frontal cortex. (A)Northern blots of COX-2 and COX-1 mRNA in AD frontal cortex relative tocontrols quantitative analysis of data is in (B). (C) Western blots ofCOX-2 protein in AD frontal cortex and control; quantitative analysis ofdata is in (D).

[0024]FIG. 16. COX-2 mRNA regulation in neurons of the anterior andposterior horn of the spinal cord of ALS and neurological controls.Autoradiographic images were visualized by X-ray film. The arrow pointedtoward the ventral horn shows intense induction of COX-2 mRNA signal inthe ALS case.

[0025] FIGS. 17A-D. COX-2 mRNA elevation in a biopsy of human corticalepileptic foci as assessed by in situ hybridization assay.Autoradiographic images were visualized by X-ray film. Arrow pointedtoward the cortical layers showing intense COX-2 mRNA signal inepileptic brain (A) versus control (B). Parts (C) and (D) show analysisof compiled data relating to COX-2 and COX-1 mRNA levels.

[0026]FIG. 18. Potentiation of cyclooxygenase and peroxidase activitiesby aggregated Aβ peptides.

[0027] FIGS. 19A-B. Transient expression of COX-2 in neuronal cellspotentiates Aβ-mediated impairment of redox activity. SH-SY5Y neuronalcells were transfected with either the human COX-2 (A) or COX-1 (B) geneand treated with Aβ₂₅₋₃₅, and then redox activity was assessed by MTTassay.

[0028] FIGS. 20A-B. Generation of Transgenic Mice Expressing COX-2. (A)In situ hybridization demonstrates COX-2 mRNA expression in TgNHC32 micebut not wild-type (mice one month old). (B) Regional expression ofhCOX-2 mRNA in NHC32 and NHC5 relative to wild-type, as quantified usingBioquant image-analysis.

[0029]FIG. 21. hCOX-2 over-expression potentiates Aβ-mediated oxidativeimpairment in primary mouse neuronal cultures.

[0030]FIG. 22. Nimesulide protects B12 neuronal cells against glutamatemediated oxidative stress.

[0031]FIG. 23. Control studies for FIG. 22. Dose (A) and time course (B)association with glutamate mediated oxidative impairment as measured byLDH assay. (C) demonstrates lack of toxicity of nimesulide to B12cultures.

5. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to the use of the cyclooxygenase-2(“COX-2”) selective inhibitor, nimesulide, and structurally relatedcompounds in the prevention and/or treatment of neurodegenerativeconditions. It is based, at least in part, on the discoveries that COX-2expression increased in parallel with neuronal lesions produced bykainic acid and with induction of neuronal apoptosis in an establishedin vitro system (see Sections 6 and 8, below). Contrary to reports inthe prior art that COX-2 plays a primary role in inflammation and may beinvolved in an inflammatory component of neurodegeneration (for example,in the context of Alzheimer's Disease), it has further been discoveredthat in the human brain, COX-2 expression appears to be restricted toneurons rather than to glial cells (whereas expression in glial cellswould be expected if an inflammatory mechanism were operative: seeSection 9, below). A correlation between COX-2 expression and thecharacteristic pathological change associated with Alzheimer's Disease,amyloid plaque, has been observed (see Section 9). Further, it has beendemonstrated that nimesulide exerts a neuroprotective effect againstβ-amyloid induced cell death in neuronal cultures. In view of thesefindings, according to the invention, nimesulide and related compoundsmay be used to intervene in the process of apoptotic neuronal cell deathassociated with Alzheimer's Disease and other neurodegenerativeconditions. Without being bound to any particular theory, the action ofnimesulide and related compounds may be mediated by the reduction of theapoptotic effects of oxidative stress.

[0033] The term “nimesulide”, as used herein, refers to a compound,4-nitro-2-phenoxymethanesulfonaninide, having a structure as set forthfor Formula I below.

[0034] Compounds which are considered structurally related are compoundswhich have a similar bicyclic structure and which selectively inhibitCOX-2. For example, substituents, analogs, and enantiomers of nimesulideare considered to be structurally related compounds. As a furtherexample, a structurally related compound may compete with nimesulide forbinding to COX-2, or may bind to substantially the same location ofCOX-2, as determined crystallographically. Moreover, nimesulide,conjugated to another compound, is also considered to be a structurallyrelated compound as defined herein.

[0035] Nimesulide, or a structurally related compound, may beadministered so as to provide an effective concentration in the nervoussystem of the subject being treated. An effective concentration isdefined herein as that concentration which inhibits neuronal cell deathby at least 20 percent. In specific, nonlimiting embodiments of theinvention, the concentration of nimesulide is at least 1 nanomolar, andpreferably at least 1 micromolar in the location of neuronal cells whichare desired to be treated. Desirably, the concentration of nimesulide isless than 10⁻³-molar to avoid toxicity. In one such specific embodiment,where the neurodegenerative condition to be treated is Alzheimer'sDisease, the concentration of nimesulide in the hippocampal formation ofthe subject is at least 1 picomolar, preferably at least 1 nanomolar,and more preferably at least 1 micromolar. The corresponding serumconcentrations may be at least 10 picomolar, preferably at least 10nanomolar, and more preferably at least 10 micromolar. Equivalentamounts of structurally related compounds (adjusted to compensate fordifferences in potency) may also be used.

[0036] Nimesulide or a structurally related compound may be administeredin any manner which achieves the desired effective concentration. Forexample, suitable routes of administration include oral, intravenous,subcutaneous, intramuscular, transdermal and intrathecal routes.

[0037] Nimesulide or a structurally related compound may be comprised ina suitable pharmaceutic carrier. Formulations may provide for sustainedrelease.

[0038] For example, but not by way of limitation, nimesulide may beadministered orally at doses of 2-800 mg/day, preferably 50-400 mg/day,and most preferably 200 mg/day.

[0039] Neurodegenerative conditions which may be treated according tothe invention include, but are not limited to, Alzheimer's Disease,Parkinson's Disease, seizure-associated neurodegeneration, amyotrophiclateral sclerosis, spinal cord injury and other diseases wherein thecondition is not conventionally regarded as an inflammation-mediated orautoimmune disorder.

6. EXAMPLE Maturational Regulation and Regional Induction ofCyclooxygenase-2 in rat Brain

[0040] 6.1. Materials and Methods

[0041] Animals and excitotoxic lesions. Male adult Sprague/Dawley ratsof different ages were maintained in a controlled light and temperatureenvironment, with food and water ad libidum. In adult rats (250-300 g),hippocampal excitotoxic lesions were induced by subcutaneous injectionof kainic acid (“KA”; 10 mg/kg, Sigma). Because KA uptake is higher inyoung rats relative to adults (Berger et al., 1986, in Schwartz andBen-Ari, Advances in Experimental Medicine and Biology, Plenum, N.Y.,pp. 199-209), KA doses were adjusted to produce maximal excitotoxicitywithout reaching lethal doses (from 2 mg/kg at postnatal day P-7 to 6mg/kg at postnatal day P25). Saline injected rats were used as controls(0 hour time point).

[0042] COX-1 and COX-2 cDNA probes. Bluescript plasmid (Stratagene)containing the full length rat COX-1 cDNA (2.7 kb) was linearized bydigestion with ClaI; PCRII plasmid (Invitrogen) containing the codingsequence for rat COX-2 (1.8 kb) was linearized by digestion with PflMI(Feng et al.,1993, Arch. Biochem.

[0043] Biophys. 307:361-368). Linearized plasmids were purified usingElu-Quick (Schleicher & Schuell) after agarose gel electrophoresis.

[0044] Int situ hybridization. At various intervals after the onset ofKA-induced seizures, the rats were sacrificed, and the brains quicklyremoved, rinsed in cold phosphate buffer (PBS, 10 mM, pH 7.4) andimmersed in methylbutane at −25° C. for three minutes. The brains weresliced into 10 micrometer sections, frozen, and the resulting frozensections were mounted on polylysine-coated slides and stored at −70° C.For immunocytochemistry (“ICC”) or in situ hybridization (“ISH”), frozensections were post-fixed in PBS containing 4 percent paraformaldehyde(30 minutes at room temperature) and then rinsed in PBS. For ISH, tissuesections were rinsed in 0.1M triethanolamine (“TEA”), pH 8.0, incubatedin acetic anhydride (“AAH”; 0.25% v/v in TEA, 10 minutes) and rinsed inTEA and PBS. Following AAH treatment, tissue sections were hybridizedwith [³⁵S]-cRNA probes (0.3 μg/ml, 2×10⁹ dpm μg-⁻¹) made from COX-2linearized cDNA transcription vectors (Feng et al., 1993, Arch. Biochem.Biophys. 307:361-368). Sense strand hybridization was used as a controland gave negative results. Following hybridization for 3 hours at 50°C., stringent washes (0.1×SSC at 60° C.) and dehydration, slides wereexposed to X-ray film for seven days for quantification. Slides werethen exposed to NTB-2 emulsion (Kodak, Rochester, N.Y.) for microscopicanalysis of COX-2 mRNA distribution. Following development, tissuesections were counterstained with cresyl violet. Film autoradiogramswere analyzed quantitatively using an image analysis system withsoftware from Drexel University (Tocco et al., 1992, Eur. J. Neurosci.4:1093-1103). Statistics were calculated by ANOVA followed by additionalposthoc analysis.

[0045] In situ end labeling. In parallel studies, paraformaldehyde-fixedbrain tissue sections were dehydrated, air dried and incubated withdATP, dCTP, dGTP (0.2 mM), dTTP (13 μM), digoxigenin-11-dUTP and DNApolymerase I (Boehringer Mannheim) at 10 units/100 μl at 37° C. for 2hours. The reaction was stopped by addition of 20 mM EDTA, pH 8.0.Sections were incubated at room temperature overnight with analkaline-phosphatase-conjugated digoxigenin antibody (Genius System,Boehringer Mannheim) diluted 1:200 in 5 percent sheep serum diluted in150 mM NaCl, 100 mM TRIS-HCl, pH 7.5. Colorimetric detection with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate was performedwith the Genius System by the manufacturer's protocol (Sakhi etal.,Proc. Natl. Acad. Sci. U.S.A. 91:7525-7529).

[0046] Northern blot hybridization assay. RNA extraction was performedas follows. After sacrifice, rat brains were dissected and stored at−75° C. prior to processing. Total RNA was extracted from pools ofhippocampal tissue (Pasinetti et al.,1994, J. Comp. Neurol.339:387-400). Briefly, tissues were homogenized for 1 minute in 4 Mguanidinium thiocyanate, 25 mM sodium citrate (pH 7.5), 0.5% sarcosyland 0.1 M β-mercaptoethanol, in a final volume of 0.5 ml. Afteracidified phenol/chloroform extraction and ethanol precipitation, theRNA pellet was rinsed consecutively with 70% and 100% ethanol. Thepurified RNA was then dissolved in 0.5% sodium dodecyl sulfate (“SDS”)and stored at −75° C. Total RNA was quantified in a UVspectrophotometer. Total RNA (5 μg) from tissue was electrophoresed ondenaturing (0.2M formaldehyde) agarose gels and transferred to a nylonmembrane (Nylon 66 plus; Hoeffer, San Francisco Calif.) in 2×SSC. Blothybridization was carried out with 10⁶ cpm/ml of antisense COX-1 orCOX-2 [³²P]-cRNA probes in 50% formamide, 1.5×SSPE, 1% SDS, 0.5% drymilk, 100 μg/ml yeast total RNA and 500 mg/ml salmon sperm DNA at 53° C.for about 15 hours. Blots were washed to a final stringency of 0.2×SSC,0.2% SDS at 72° C. Blots were exposed to Kodak X-ray film (XAR-5) withintensifying screens at −70° C.

[0047] Cell cultures. Hippocampal neuron cultures were derived fromembryonic rat brain. E16-E18 embryos were dissected in Hank's balancedsalt solution and cultured (Pasinetti et al.,1994, J. Comp. Neurol.339:387-400; Peterson et al., 1989, Dev. Brain Res. 48:187-195). Culturemedia were changed every 3 days. For glutamate neurotoxicity studies,eight day old cultures were treated with glutamate (250 μM, Sigma) inthe presence of 2.4 mM calcium ion and 0.8 mM magnesium ion. After 6hours of glutamate exposure, culture medium was replaced with freshmedium; COX-2-like immunoreactivity was assessed in neurons 12 hourslater.

[0048] Immunocytochemical detection of COX-2 in monotypic cultures ofprimary rat neurons. Control and glutamate treated cultures werepost-fixed in PBS containing 4% paraformaldehyde (30 minutes, roomtemperature), rinsed in PBS, pre-treated with normal serum and incubatedovernight at 4° C. with primary antibodies. COX-2 antisera (rabbit IgG)was raised against a synthetic peptide (CNASASHSRLDDINPT; SEQ ID NO:1)encompassing the C-terminal region of the murine COX-2. The antiserareacts with human and rat COX-2 but not with COX-1, as assessed byWestern blot analysis. Vectastain ABC kit (Vector, Burlingame, Calif.)was used in subsequent steps to complete the diaminobenzene staining(Pasinetti et al.,1994, J. Comp. Neurol. 339:387-400). Immunoadsorptionof COX-2 antisera with synthetic COX-2 peptides controlled forspecificity; adsorption was carried out overnight at 4° C. withsynthetic COX-2 peptides at 30 μg/ml.

[0049] 6.2. Results

[0050] COX-2 expression in adult rat brain. FIG. 1 shows by ISH that theregional distribution of COX-2 mRNA was most notable in limbicstructures, but was also present in neocortex (consistent with thereports of Kaufmann et al., Proc. Natl. Acad. Sci. U.S.A. 93:2317-2321and Yamagata et al., 1993, Neuron 11:371-386). In the hippocampalformation, COX-2 mRNA was selectively expressed in cells of the granuleand pyramidal neuron layers. COX-2 mRNA expression was also found in theouter layer of the parietal cortex, the pyriform cortex and cells of theamygdaloid complex.

[0051] Maturational regulation of hippocampal COX-2 mRNA expression. ISHresults indicated that during maturation, COX-2 MnRNA expression isdifferentially regulated in subsets of cells of neuronal layers of thehippocampal formation (FIG. 2, FIG. 3 top). From postnatal days P7-P14,COX-2 mRNA showed greater than two-fold increase in the granule celllayer of the dentate gyrus and in the CA3 subdivision of the pyramidalcell layer (p<0.001, FIG. 2). Though the expression of COX-2 mRNA waslower in the other brain regions examined, the pattern of maturationalexpression was similar (FIG. 2). By postnatal day P21, COX-2 mRNAexpression approximated adult levels in all subregions examined (FIG. 2,FIG. 3 top).

[0052] Response to KA-induced seizures. To further explore maturationalregulation of COX-2 in brain, COX-2 mRNA expression during responses toKA-induced seizures was examined postnatally. Despite intense seizureactivity after KA treatment, no detectable change of COX-2 mRNAexpression was found in any brain region examined at P7 (FIG. 3, FIG.4A). Changes in COX-2 mRNA expression at 120 hours post-KA treatment inthe P7 group indicate developmental maturation rather than response toKA toxicity (FIG. 4A). In contrast to the P7 group, at P14 and P21 COX-2mRNA increased within 4-8 hours after onset of KA-induced seizures inall the hippocampal subregions examined (FIGS. 4B and 4C). The level ofCOX-2 mRNA expression returned toward control levels within 120 hoursafter treatment in P14, P21 and adult rat brain.

[0053] Within the dentate gyrus, control COX-2 expression in P14 and P21rats was asymmetric, selectively localized to the more superficialneurons of the stratum granulosum rather than the deeper granule cellsof the dentate gyrus blade (FIG. 5A). In response to KA treatment, COX-2mRNA induction showed similar asymmetry of expression (FIG. 5B). Incontrast, the asymmetry within the dentate gyrus was less notable incontrol animals and after KA-induction of the adult group (FIGS. 5C and5D).

[0054] In parallel studies, northern blot hybridization of total RNAfrom hippocampus of adult rats 12 hours after KA-induced seizuresconfirmed COX-2 mRNA induction (FIG. 6). No detectable induction ofCOX-1 mRNA was found in the same rat brain (FIG. 6).

[0055] KA-induced COX-2 and apoptosis in adult rat. By 8 hours afteronset of KA-induced seizures, COX-2 mRNA induction in cells of the CA3region of the hippocampal formation (FIG. 7B), pyriform cortex (FIG. 7E)and amygdaloid complex (FIG. 7H) of the adult brain paralleledtemporally and overlapped anatomically the onset of apoptosis asassessed by in situ end-labeling in the same brain regions (FIG. 7C, CA3regions of the hippocampus; FIG. 7F, pyriform cortex; FIG. 7I,amygdaloid complex). Cellular COX-2 mRNA expression in adult, control(FIGS. 7A, 7D and 7G) and KA-treated (FIGS. 7B, 7E and 7H) rats wasidentified by emulsion autoradiography using ISH assays.

[0056] Immunocytochemical evidence of neuronal COX-2expression/regulation in response to glutamate in vitro. Primarycultures of rat hippocampal neurons were exposed to glutamate in vitro.At baseline, constitutive COX-2 expression was demonstrated byimmunocytochemistry (FIG. 8B). Twelve hours after exposure to glutamate,an increase in COX-2 immunoreactivity was observed which coincided withmarked reduction in the number of neurons (FIG. 8D).

[0057] COX-2 expression in human epilepsy. Increased expression of COX-2mRNA was detected by ISH in a biopsy of human brain at epileptic foci(FIGS. 17A-D). It was found that COX-2 mRNA, but not COX-1 mRNA, wassignificantly elevated in the cortex of epileptic patients. the p vlauewas <0.05.

7. EXAMPLE Nimesulide Suppresses Cytokine and Nitrite Production inMicroglia Cultures

[0058] In experiments conducted in vitro, nimesulide at lowconcentration (1 nanomolar) was found to be effective in the suppressionof endotoxin-mediated induction of tumor necrosis factor (“TNF”)production by immortalized brain-derived microglia (BV-2) and astrocytes(FIG. 9A). In parallel, nimesulide was equally effective in blockingnitrite production (Griess reaction; FIG. 9B). This latter observationis particularly relevant in view of evidence showing that blockade ofneuronal nitric oxide (“NO”)-synthase protects against glutamateneurotoxicity. Nimesulide was also found to be effective in blockingendotoxin-mediated induction of prostaglandin PGE₂) in brain-derivedmicroglia (FIG. 9C).

8. EXAMPLE COX-2 Expression in Apoptotic Cells

[0059] The regulation of COX-2 expression was studied using anestablished in vitro model of apoptosis. Specifically, the regulation ofCOX-2 was studied in P19 embryonic carcinoma cells during responses toserum deprivation. Under such conditions, the P19 cells underwentapoptotic cell death, showing characteristic DNA fragmentation andnuclear morphology. As shown in FIG. 10, using this system, coincidentalonset of apoptosis and elevation of COX-2 expression was observed.

[0060] These studies were extended to the human neuronal cell lineSH-SY5Y. β-amyloid has been demonstrated to play a role in inducingneurodegeneration in SH-SY5Y cells (Oda et al., 1995, Alzheimers Res.1:29-34). SH-SY5Y cells were treated with synthetic aggregated Aβ₁₋₄₀peptides at a concentration of 20 μM for various periods of time.Western blot analysis was performed of cell extracts, using COX-2antisera or actin antisera as control (FIGS. 11B and D, respectively).In FIG. 11A, COX-2 protein from control or Aβ-treated SH-Sy5Y neuronalcells was quantified from the immunoblots shown in FIG. 11B (at the 72hr. time point; only the 70 kDa mw species being quantified). COX-2signal was abolished by immunoadsorption of COX-2 antisera with purifiedhuman recombinant COX-2 (“hrCOX-2”) peptides. FIG. 11C shows thatdiminished cell redox activity was observed 72 hours after treatmentwith aggregated Aβ₁₋₄₀ (at 20 μm), as assessed by MTT assay in parallelcultures. Bar graphs represent the mean±SEM, n=4-5 per group, p<0.05(t-test). COX-2 integrated optical densities were analyzed fromdigitized images using a Bioquant image analysis (Biometrics, Nashville,Tenn.). It was also found that COX-2 expression occurred in parallel toDNA laddering (FIG. 11E), an index of apoptosis. FIG. 12 shows thatnimesulide at 10⁻⁶ and 10⁻⁹ molar was able to block Aβ1-40 toxicity, asassessed by the MTT assay.

[0061] Interestingly, when aggregated synthetic Aβ₁₋₄₀ peptides (150 μM)were coincubated with purified hr-holoCOX-2 (COX-2 plus heme-cofactor,50 nM) for 16 hours, at 37° C. in 1X PBS, and cyclooxygenase andperoxidase activities were measured (Murphy et al., 1989, Neuron2:1547-1558) cyclooxygenase and peroxidase activities increased(relative to activity levels of enzyme in the absence of Aβ; FIG. 18).Cyclooxygenase and peroxidase levels in the absence of heme gavenegative results.

9. EXAMPLE COX-2 Expression in Alzheimer's Disease

[0062] The expression of COX-2 in normal human brains and in brains ofAlzheimer's Disease (“AD”) patients was studied. Immunocytochemical dataindicates that COX-2 expression is primarily localized to cells withneuronal morphology in human brain; minimal localization of COX-2immunostaining in cells with glial morphology was found in any regionexamined. These results tend to suggest a role for COX-2 in anon-inflammatory function. While the immunocytochemical signal for COX-2was at the limit of detection in temporal cortex of neurological controlcases, COX-2 showed elevation in AD brain (FIG. 13A, 13B) The selectiveinduction of COX-2 immunoreactivity in grey matter is consistent withthe findings showing neuronal induction of COX-2 in rat brain duringresponses to lesions leading to neuronal death (see Section 6).

[0063] The cellular immunodistribution of COX-2 in AD brain was alsoexplored. It was observed that neuronal COX-2 is not only localized tothe perikarya (FIG. 14A, but is found in neuronal projections as well(FIG. 14B). Moreover, intense immunostaining was also found in diffuseplaques (FIG. 14C) and in AD neuritic plaques identified by Aβimmunostaining on adjacent tissue sections of the hippocampal formation.These findings suggest a role for COX-2 in mechanisms of neuronal deathor survival.

[0064] Studies were performed to quantify COX-2 expression in brains ofAD and age-matched controls. We used quantitative dot-blot analysis andchemiluminescence detection. Western analysis was used for qualitativeassessment. A greater than 2-fold elevation of COX-2 content was foundin hippocampal homogenates of AD brains compared to neurologicalage-matched controls.

[0065] Because of the immunocytochemical evidence showing COX-2immunoreactivity in AD plaques, levels of COX-2 and total Aβ in AD caseswere compared. A direct correlation was found.

[0066] COX-2 mRNA and protein were found to be increased in AD brainfrontal cortex. FIG. 15A shows Northern blots of total RNA fromneurological control (C) and AD frontal cortex hybridized to [32p] COX-2and COX-1 mRNAs. The mRNA species detected by COX-2 and COX-1 cRNAprobes represent an RNA of the same size found in peripheral tissues.FIG. 15B shows quantified results of the Northern studies. Specifically,COX-2 mRNA prevalence was increased in the frontal cortex of AD vscontrol, where the presence of equal amounts of RNA in both lanes wasconfirmed by normalization to 28S rRNA in the hybridization membrane,and complete transfer of RNA to the membrane was evidenced by theabsence of 28S and 18S rRNAs in the gel post transfer. Integratedoptical densities were analyzed from digitized images using a Biquantimage analysis. Bar graphs represent n=9 per group, and the results weresignificant by a t-test value of p<0.05.

[0067] Similarly, when Western blot analysis was performed of AD frontalcortex vs. controls, the amount of COX-2 protein was found to increase.FIG. 15C shows the results of such a western blot. Lanes 1 and 2 showthe COX-2 protein content of the same tissues uses for Northernanalysis. Lanes 3-4 show the absence of COX-2 bound when COX-2 antiserawas immunoadsorbed using hrCOX-2 peptides, demonstrating the specificityof the signal.

[0068] The expected 70 kDa new species of hrCOX-2 (lane 5) also wasabsent when antisera was preadsorbed with hr COX-2 peptides. Theseresults are quantified in FIG. 15D (with respect to the 70 kDa species).Further, the immunoblots were stripped and imnnunoreacted with actinantisera to verify the specificity of changes (actin values: controlgroup 100±8; AD 94±7% of control Bar graphs represent n=9 per group;t-test value is p<0.05). The inset in FIG. 15D shows a correlation ofCOX-2 content and the number of plaques per mm², n=8, r=0.72, p<0.03.COX-2 and actin integrated optical densities were analyzed fromdigititzed images using Biquant image analysis.

10. EXAMPLE COX-2 Expression in Amyotrophic Lateral Sclerosis

[0069] In situ hybridization has demonstrated increased COX-2 mRNA inthe anterior horn cells of spinal cords of patients suffering fromamyotrophic lateral sclerosis (“SACS”); FIG. 16.

11. EXAMPLE Transient Expression of COX-2 In Neuronal Cells PotentiatesAβ Mediated Impairment of Redox Activity

[0070] The role of COX-2 in neuronal death and/or survival was studiedin cultured human SH-SY5Y neuronal cells. Cells were transientlytransfected with a mammalian expression vector (pRc/CMV/hCOX,Invitrogen) containing either a filll length human (h) COX-2 cDNA(pRc/CMV/hCOX-2), or a bacterial chloramphenicol acetyltransferase (CAT)gene (pRc/CMV2/CAT). Following Aβ25-35 treatment, impairment of redoxactivity as assessed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetraxolium bromide (MTT)-assay(FIG. 19). Overexpression of hCOX2 in SH-SY5Y cells transfected withpRc/CMV/hCOX-2 was confirmed immunocytochemically in a parallel tissueculture chamber slide using an α-COX-2 antibody as previously described.In control cells transfected with pRc/CMV2/CAT, the same α-COX antibodygenerated minimal immunocytochemical signal. We found that transientoverexpression of hCOX-2 in SH-SY5Y neuronal cells potentiated Aβ25-35(25 μM, 48 hrs treatment) mediated impairment of redox activity whencompared to SH-SY5Y cultures transfected with control vector (±±p<0.01vs Aβ/control vector; *p<0.001 vs CTL. These data are consistent withevidence showing induction of COX-2 mRNA coincidentally with impairmentof redox activity in SH-SY5Y neuronal cells (FIG. 11).

12. EXAMPLE Experiments Using COX-2 Transgenic Mice

[0071] Based on the evidence that overexpression of COX-2 in neuronalcells may potentiate Aβ -mediated redox impairment and the observationthat neuronal COX-2 is elevated in AD brain and in experimentalneurodegeneration, we generated a transgenic mouse model with neuronaloverexpression of human (h) COX-2. To obtain overexpression of hCOX-2 inneurons, a hybrid gene was prepared in which the expression of a cDNAsequence containing the entire hCOX-2 coding region is regulated by therat neuron-specific enolase (NSE) promoter.

[0072] We obtained four lines with high expression of hCOX-2 mRNAexpression. In one of them (NHC32), COX-2 mRNA was assessed by in situhybridization analysis (FIG. 20A). We found that in NHC32 mouse line,has high levels of hCOX-2 mRNA in the hippocampal formation, cerebralcortex and in other neuronal layers (FIG. 20B). No white matter level ofHCOX-2 mRNA were found using combined immunocytochemistry for NSE andhCOX-2 in situ hybridization on the same tissue section, we confirmedthe selectivity of hCOX-2 transgene expression to neuronal cells.

[0073] It has further been shown, using neuronal cultures derived fromthe transgenic mice, that hCOX-2 overexpression potentiated Aβ mediatedresponse. As depicted in FIG. 21, studies showed that primary neuronalcultures derived from transgenic mice with neuronal COX-2 overexpression(NHC32, n=3), are most susceptible to aggregated Aβ25-35 peptidesmediated impairment of redox activity (MTT assay, Aβ25-35 25 μM for 48hrs), when compared to equally treated neuronal cultures derived fromwild type/control littermates (n=4). Moreover, when examinedmorphologically, Aβ25-35 treated neurons derived from transgenic hCOX-2mice revealed an intensified regression of neuronal processes (B, inset)when compared to wild type control Aβ25-35 treated neuronal cultures (A,inset).

13. EXAMPLE Nimesulide Protects B12 Neuronal Cells Against GlutamateToxicity

[0074] We examined the role of nimesulide, indomethacin and NS398(another COX-2 preferential inhibitor) on glutamate mediated toxicity.In this study we used B12 neuronal cells; toxicity was assessed byamount increase of lactate dehydrogenase LDH levels in the conditionedmedium after treatment. LDH is an index of cell toxicity and itselevation is generally accepted as marker of neurotoxicity. As shown inFIGS. 22-23, we found that nimesulide protected against glutamatetoxicity (10 mM) at 10−6, 10−9 and 10-12 M with pretreatment (24 hrsbefore) and cotreatment (with glutamate for 24 hrs). Indomethacin,protected at 10−6 M and lost its protective effect at 10−9 and 10-12 M.Marginal protection of glutamate mediated toxicity by indomethacin wasobserved at 10−9 M (pretreatment 24 hrs). No protection was observedwith NS398 pretreatment. In the pretreatment condition drugs remained inthe media during glutamate treatment.

[0075] Various publications are cited herein, the contents of which arehereby incorporated in their entireties.

1 1 1 16 PRT Artificial Sequence synthetic peptide 1 Cys Asn Ala Ser AlaSer His Ser Arg Leu Asp Asp Ile Asn Pro Thr 1 5 10 15

What is claimed is:
 1. Use of nimesulide for the preparation of apharmaceutical composition for treating a neurodegenerative condition.2. Use of nimesulide for the preparation of a pharmaceutical compositionfor treating Alzheimer's Disease.
 3. Use of nimesulide for thepreparation of a pharmaceutical composition for treating epilepsy. 4.Use of nimesulide for the preparation of a pharmaceutical compositionfor treating amyotrophic lateral sclerosis.
 5. Use of nimesulide for thepreparation of a pharmaceutical composition for treating aneurodegenerative condition in a subject, wherein the amount ofnimesulide present in the pharmaceutical composition results in exposureof the affected neurons to a local concentration of nimesulide of onemicromolar or a lower concentratioin that inibits neuronal cell death byat least 20 percent.
 6. Use of nimesulide for the prepararion of apharmaceutical composition for treating Alzheimer's Disease in asubject, wherein the amount of nimesulide present in the pharmaceuticalcomposition results in exposure of the affected neurons to a localconcentration of nimesulide of one micromolar or a lower concentrationthat inhibits neuronal cell death by at least 20 percent.
 7. Use ofnimesulide for the preparation of a pharmaceutical composition fortreating epilepsy in a subject, wherein the amount of nimesulide presentin the pharmaceutical composition results in exposure of the affectedneurons to a local concentration of nimessulide of one micromolar or alower concentration that inhibits neuronal cell death by at least 20percent.
 8. Use of nimesulide for the preparation of a pharmaceuticalcomposition for treating amyotrophic lateral sclerosis in a subject,wherein the amount of nimesulide present in the pharmaceuticalcomposition results in exposure of the affected neurons to a localconcentration of nimesuide of one micromolar or a lower concentrationthat inhibits neuronal cell death by at least 20 percent.
 9. Atransgenic mouse carrying a gene encoding the human cyclcoxygenase-2gene under the control of a neuron-specific promoter.
 10. The transgenicmouse of claim 9 wherein the level of cyclooxygenase-2 activity presentin some or all neurons of the mouse is increased.
 11. A neuronal cellculture prepared from the transgenic mouse of claim 9 or 10).
 12. Use ofnimesulide for the preparation of a pharmaceutical composition fortreating a neurodegenerative condition, wherein the dosage of nimesulidecontained in the pharmaceutical composition inhibits neuronal cell deathby at least 20 percent.
 13. Use of nimesulide for the preparation of apharmaceutical composition for treating Alzheimer's Disease, wherein thcdosage of nimesulide contained in the pharmaceutical compositioninhibits neuronal cell death by at least 20 percent.
 14. Use ofnimesulide for the preparation of a pharmaceutical composition fortreating epilepsy, wherein the dosage of nimesulide contained in thepharmaceutical composition inhibits neuronal cell death by at least 20percent.
 15. Use of nimesulide for the preparation of a pharmaceuticalcomposition for treating amyotrophic lateral sclerosis, wherein thedosage of nimesulide contained in the pharmaceutical compositioninhibits neuronal cell death by at least 20 percent.